The goal of this research proposal is to define the biochemical mechanisms governing the interaction of HIV RT with its replication primer, tRNALys,3. In a multidisciplinary approach, we intend to evaluate the possibility of inhibiting this interaction, which might allow therapeutic intervention at initiation of viral replication, as opposed to agents which block an actively synthesizing enzyme. RT/tRNA interactions will use several forms of highly purified recombinant enzyme, together with both natural and in vitro-synthesized forms of tRNA. Using synthetic tRNA, we can evaluate the contribution of individual bases or entire domains to both specificity and affinity of this interaction. Once these models are established, we will study the tRNA sequestering capacity of precursor, RT-containing polyproteins. Together with these studies, a program is presented to elucidate the tRNA binding domain(s) of RT, after which we will alter important amino acids within this and determine their contribution to primer binding. In a later program of "subunit-selective mutagenesis", we will evaluate the properties of a reconstituted heterodimer RT, within which one subunit has been altered in its polymerizing or tRNA binding domain. Experiments on primer binding will be extended to study "arrested" transcriptional complexes, to monitor interactions between RT and its primer/template. Finally, it is planned to use data accumulated on this RNA/protein interaction in a biophysical program. Both x-ray crystallography and low angle neutron scattering will be used to gain structural information on subunit arrangement within the heterodimer, as well as conformational changes in RT/primer/template complexes.